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Culturing Methods and Information

NCMA Definition of Terms


Scientific name: We rely on the depositor and/or the phycological community to identify algal strains deposited at the NCMA.  We do not routinely do DNA analysis, pigment analysis, or use other methods to identify algal strains or to verify the claims of a depositor. However, we do microscopic examinations to confirm that strains resemble the species that they are claimed to be.  

Strain Synonyms: These are non-CCMP numbers or names that others have assigned to this strain (such as the depositor, or a different algal collection). Entries in this field are not necessarily inclusive of all strain aliases assigned to a specific strain.

Maintenance temperature: The maintenance temperature is the temperature at which we maintain a strain at the NCMA.

Know temperature range (should read reported): The known (reported) temperature range of a strain is the information we have gathered from our observations growing the strain at the NCMA, information in the literature, or hearsay information.  These ranges are not necessarily used for long term maintenance of a particular strain. We can only vouch for successful maintenance of a strain at the temperature at which we maintain it at the NCMA.

Size information: This information results from an examination of a strain with a compound microscope as an isolated event. The size of individual cells of many algal species can change over time.

Toxic: A strain is defined as toxic if the species is known to produce toxins or if a strain with an undefined species name is a member of a genus that is known to have toxin producing members. We do no toxin testing on site.

Depositor: The depositor is the individual who sent the strain to the NCMA

Isolator: The isolator is the individual(s) who isolated the strain from a raw sample

Collector: The collector is the individual(s) who collected the water sample from which a strain was isolated

 

 Culture Medium Types

Enrichment media and artificial media are the two major types of culture media. An enrichment medium is usually prepared either (1) by adding soil or soil extracts to distilled water or natural water or (2) by adding nutrient chemicals to natural water (e.g., seawater or lake water). An artificial medium uses "pure" water and "pure" chemicals only; it does not include additions of undefined soil or natural lake or sea water. However, it is very important to recognize that unknown "impurities" are present in even the most carefully prepared artificial medium.

Soil-water enriched media are often best for maintenance of algal cultures. Good soils contribute inorganic and organic materials - excellent algal growth occurs, and culture induced morphological changes are limited.

Marine media prepared by chemically enriching seawater are common. However, the use of lake or river water for chemically enriched freshwater cultures is uncommon. Conversely, artificial media are common and very successful for the growth of many freshwater algae, but marine artificial media are used only when critical studies cannot be conducted using a natural seawater base. For example, a carefully defined, artificial seawater medium is used to minimize or exclude known contaminants for the purpose of studying trace elements.

Similarly, water quality, glass/plasticware cleanliness, etc., impact a culture medium in the same manner as chemical quality, i.e., the presence of undesirable or toxic materials and the absence of required substances affect algal growth. Culture media and culturing vessels can be sterilized by steam autoclaving, pasteurization, filtration or microwaves.


Culturing Vessels and Materials

All containers and tubing used for growing cultures and storing culture medium stocks should be carefully selected to avoid toxic compounds. We recommend flasks and test tubes made of borosilicate glass or tissue culture-grade polycarbonate and polystyrene plasticware. Black test tube screw caps should be autoclaved several times in changes of seawater because new caps may release toxic phenolics when heated.

Likewise, rubber stoppers (or anything else that releases odorous volatile compounds when heated) should be autoclaved separately from media. Older autoclaves with copper tubing should be avoided because excess copper is toxic to algae. The autoclave steam itself may be contaminated with metals, and a culture medium may become metal-toxic to open ocean species.

New glassware should be degreased in dilute NaOH. Glassware may be routinely cleaned in dilute HCl or, if metal contamination is a concern, a long soak in concentrated HCl is recommended. Glassware should not be cleaned in chromic acid because chromium is toxic to many phytoplankters. Flasks may be capped with plugs made of cotton wrapped in cheesecloth, silicone sponge or covered with a beaker. An over-cap made of foil or a plastic beaker may prevent fungi from becoming established in a damp plug.


Preparing Culture Media

Most scientists enrich natural seawater when preparing cultures (Seawater can be purchased from the NCMA. The source of seawater may influence success. Some coastal waters have reduced salinities that may not be tolerated by some strains. The NCMA uses pristine water from the Gulf of Maine, which has a salinity of approximately 32 psu. Seawater should not be collected during blooms, especially when noxious organisms are present. Natural phytoplankton can be removed using filters (e.g., 0.45µm glass fiber filter). Dissolved organic contaminants can be removed by adding one to several grams of activated carbon powder per liter and mixing thoroughly. The carbon is removed the following day by decanting and filtering.

To simplify routine medium preparation, working stock solutions are usually prepared. Adding a small volume of a liquid stock solution is easier and quicker than weighing individual dry chemicals. It is better to add stocks to water and mix thoroughly - direct combinations of stock solutions without dilution in water may result in undesirable precipitation. Nutrient enrichments are either added prior to sterilization or added aseptically after sterilization. Sterilization is accomplished by autoclaving at 121°C, 15 lb/in2 for 15 minutes or longer depending on the volume involved. The culture medium should then be cooled as rapidly as possible after autoclaving to avoid precipitation. Silica enhances precipitation, and therefore if silicon is not required by the alga, it is better to leave silicates out of the medium.

Nutrient stocks may become contaminated with a bacterium or fungus. It is prudent, therefore, to initially autoclave stock solutions and then practice good sterile technique. Stock solutions should be carefully sealed because evaporation will concentrate nutrient stocks. Vitamin stocks may be frozen for long periods of time without noticeable degradation. Autoclaving the Na2SiO3 stock solution in a glass container may result in etching or shard-like precipitation of silica. Therefore we recommend preparing the silicate stock solution in a teflon-coated bottle.

After sterilization by autoclaving, the culture medium should set for about 24 hours while gases (especially CO2) diffuse into the liquid. Precipitation is minimized by rapid cooling. A final pH of 7.8-8.2 is desirable for most seawater media.

General References

Berges, J.A., Franklin, D.J. and Harrison, P.J. 2001. Evolution of an artificial seawater medium: improvements in enriched seawater, artificial water over the last two decades. J. Phycol. 37:1138-45.

Bernhard, M. and Zattera, A. 1970. The importance of avoiding chemical contamination for a successful cultivation of marine organisms. Helgolander wiss Meeresunters 20: 655-675.

Bernhard, M. 1977. Chemical contamination of culture media: assessment, avoidance and control. pp. 1459-99. In Kinne, O. (Ed.) Marine Ecology. Vol III. Cultivation Part 3. Wiley & Sons, Chichester.

Blankley, W. 1973. Toxic and inhibitory materials associated with culturing. pp. 207-229. In Stein, J. (Ed) Culture Methods and Growth Measurements. Cambridge University Press, Cambridge, U.K.

Fogg, G.E. 1965. Algal Cultures and Phytoplankton Ecology. Univ. of Wisconsin Press, Madison, 126p.

Guillard, R.R.L. 1975. Culture of phytoplankton for feeding marine invertebrates. pp 26-60. In Smith, W.L. and Chanley, M.H. (Eds) Culture of Marine Invertebrate Animals, Plenum Press, New York.

Harrison, P.J., Waters, R.E. and Taylor, F.J.R. 1980. A broad spectrum artificial seawater medium for coastal and open ocean phytoplankton, J. Phycol 16: 28-35.

Hellbust, J.A. and Craigie, J.S. (Eds) 1978. Physiological and Biochemical Methods. Cambridge Univ. Press, Cambridge, U.K. 512 pp.

Keller, M.D., Selvin, R.C., Claus, W. and Guillard, R.R.L. 1987. Media for the culture of oceanic ultraphytoplankton. J. Phycol. 23: 633-638.

Lehman, T.L. 1976. Ecological and nutritional studies on Dinobryon Ehrenb.: seasonal periodicity and the phosphate toxicity problem. Limnol. Oceangr. 21: 646-658.

Price, N.M., Harrison, G.I., Hering, J.G., Hudson, R.J., Nirel, P.M.V., Palenik, B. and Morel, F.M.M. 1988/89. Preparation and chemistry of the artificial culture medium Aquil. Biol. Oceanogr. 6: 443-461.

Price, N.M., Harrison, P., Landry, M.R., Azam, F. and Hall, K. 1986. Toxic effects of latex and Tygon tubing on marine phytoplankton, zooplankton and bacteria. Mar. Ecol. Prog. Ser. 34: 41-49.

Pringsheim, E.G. 1946. Pure Cultures of Algae. Their Preparation and Maintenance. Cambridge Univ. Press, Cambridge, U.K. 119 pp.

Provasoli, L. 1971. Media and prospects for the cultivation of marine algae. pp. 63-75. In Watanabe, A. and Hattori, A. (Eds) Cultures and Culture Collections. Jap. Soc. Plant Physiol.

Richmond, A. (Ed) 1986, CRC Handbook of Microalgal Mass Culture, CRC Press, Boca Raton, Florida. 528 pp.

Stein, J.R., (Ed) 1973. Culture Methods & Growth Measurements. Cambridge University Press, Cambridge, U.K. 448 pp.

Suttle, C., Price, N., Harrison, P. and Thompson, P. 1986. Polymerization of silica in acidic solutions: a note of caution to phycologists. J. Phycol. 22: 234-237.

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